This invention relates to an improved method for identifying polarizable particles, macromolecules, and biological cells and a device for carrying out the method.
Identification of molecules, particles, and biological cells constitutes a major part to their purification, utility, and production. Identifying components in solution is equally important for both the biotechnology and chemical industries. In the chemical polymerization to manufacture commercial polymers and latex particles, for example, knowledge and control of the distribution of particle growth and size are critical. The situation is at least as complicated for biological organisms, where details about protein and cell populations are critical. The terms: particles, cells, macromolecules, particulates, and polymers are used here interchangeably, unless otherwise noted.
The present invention describes a device and a method for the identification of polarizable particles, macromolecules, and biological cells. The invention detects the behavior of dielectric materials in non-uniform fields. Dielectric properties are indicative of the polarizabilities of the macromolecules, cells, and particles. The detection of these properties is accomplished by measuring the modulation in the time autocorrelation function, measured by a technique such as dynamic light scattering, DLS. Current applications of DLS measure the Brownian (random) motion of molecules. Under the influence of a non-uniform electric field, polarizable macromolecules undergo characteristic motion, called dielectrophoresis. For the sake of clarity, a brief introduction to the basis of DLS and dielectrophoresis phenomena is presented below.
In a DLS experiment, a laser light impinges on a solution of macromolecules and the intensity of the scattered light is measured at an angle, .theta.. A typical apparatus setup for DLS is presented in FIG. 1. The frequency of light is Doppler shifted due to the Brownian motion of the scattering macromolecules in the scattering volume, defined by the incident and scattered beam geometries at their intersection. The frequency shifts are related to the diffusion coefficients of the scattering species. Current DLS experiments measure the Fourier transform of these frequency shifts as the time autocorrelation function of the intensity fluctuations produced by molecular motion in solution. Time autocorrelation functions are exponential with time constants which are characteristic of the diffusion coefficients of the scattering species. From these coefficients, useful information can be obtained on the scattering molecules, primarily a measure of their size in solution. For a monodisperse system of particles, the heterodyne intensity autocorrelation function, C(.tau.), can be written as: ##EQU1##
where &lt;N&gt; is the average number of particles, .tau. is the delay time used to construct C(.tau.), and q is an experimental constant wherein: ##EQU2##
Here, n is the refractive index of the solution, .lambda. is the wavelength of light and .theta. is the scattering angle. In Equation (1), D is the diffusion coefficient, which for a spherical particle is: ##EQU3##
where k is the Boltzmann constant, T is the absolute temperature, .eta. is the viscosity and r is the particle radius.
An analysis of the autocorrelation functions using Equations 1-3 can lead to the extraction of the diffusion coefficient of the scattering species and, hence, a measure of their size.
In polydisperse systems, the measured autocorrelation function is a sum of exponentials (or, for continuous distribution, an integral) representing the different species present, EQU C(.tau.)=.intg.G(.GAMMA.)e.sup.-.GAMMA..tau. d.GAMMA., (4)
where .GAMMA.is the exponent in Equation 1, (.i.e..GAMMA.=q.sup.2 D).
As Equation 4 shows, except for a single solute (monodisperse) systems, C(.tau.) data will be composed of a sum of exponentials. Analysis of multiple exponentials is difficult, even though clever methods have been developed for such analyses. The origin of the difficulty is that exponentials overlap quite strongly, with no discernible structure developed in the resulting functional form. The present invention, in addition to furnishing a new tool for the identification of macromolecules, also provides a guide to overcoming analysis difficulties by giving an estimate of the number of components present.